BACKGROUND: The low immune response to DNA vaccines remains a challenge to their potential clinical use, and the development of new delivery strategies continues to be an area of significant effort. One clinically utilized strategy to enhance the potency of protein and polysaccharide vaccines is to deliver the antigens via bacterial "outer membrane vesicles", or OMVs, which are vesicles constitutively produced by gram-negative bacteria. OMVs are used by bacteria in nature as protein, and potentially DNA, delivery vehicles. The objective of this R21 exploratory research grant is to definitively determine if OMVs can be engineered to deliver plasmid-based DNA vaccines to mammalian antigen presenting cells. HYPOTHESIS: OMV characteristics can be engineered to enhance the delivery of plasmid-based DNA vaccines to mammalian antigen presenting cells. SPECIFIC AIMS: 1) To engineer, and quantitatively evaluate, the surface of plasmid-containing OMVs to maximize their non-specific binding to mammalian cell membranes, and 2) To engineer, and quantitatively evaluate, the surface of plasmid-containing OMVs to facilitate their release from acidic subcellular vesicles. The zeta potential of OMVs is approximately -40 mV which reduces their binding affinity to mammalian cell membranes. To increase the association of OMVs with mammalian cells, and increase the rate of internalization by non- specific endocytosis or phagocytosis, the surface of the OMVs will be engineered to possess sequences of polylysine to create OMVs with positive zeta potentials. Internalization of macromolecular structures by endocytosis/phagocytosis leads to their sequestration within acidic compartments and digestion by lysosomal enzymes. To facilitate the escape of OMVs from the lysosomal trafficking pathway, the surface of the OMVs will be engineered to possess sequences of polyhistidine to disrupt the membrane of acidic vesicles. Validation of the hypothesis will support the future engineering of OMVs with more intricate characteristics, such as cell specific ligands, fusogenic proteins and conformational protein adjuvants. [unreadable] [unreadable] [unreadable]